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  • Developmental Biology 334 (2009) 31–45

    Contents lists available at ScienceDirect

    Developmental Biology

    j ourna l homepage: www.e lsev ie lopmenta lb io logy

    Spatial and temporal regulation of Wnt/β-catenin signaling is essential for development of the retinal pigment epithelium

    Naoko Fujimura a, Makoto M. Taketo b, Mikiro Mori c, Vladimir Korinek a, Zbynek Kozmik a,⁎ a Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic b Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan c Department of Ophthalmology, Jichi Medical School, Minami-kawachi, Tochigi, 329-0498, Japan

    ⁎ Corresponding author. Fax: +420 241063125. E-mail address: (Z. Kozmik).

    0012-1606/$ – see front matter © 2009 Elsevier Inc. Al doi:10.1016/j.ydbio.2009.07.002

    a b s t r a c t

    a r t i c l e i n f o

    Article history: Received for publication 21 January 2009 Revised 30 June 2009 Accepted 1 July 2009 Available online 9 July 2009

    Keywords: Wnt/β-catenin signaling Retinal pigment epithelium Neural retina Mitf Otx2

    Wnt/β-catenin signaling is highly active in the dorsal retinal pigment epithelium (RPE) during eye development. To study the role of Wnt/β-catenin signaling in the RPE development we used a conditional Cre/loxP system in mice to inactivate or ectopically activate Wnt/β-catenin signaling in the RPE. Inactivation of Wnt/β-catenin signaling results in transdifferentiation of RPE to neural retina (NR) as documented by downregulation of RPE-specific markers Mitf and Otx2 and ectopic expression of NR-specific markers Chx10 and Rx, respectively. In contrast, ectopic activation of Wnt/β-catenin signaling results in the disruption of the RPE patterning, indicating that precise spatial and temporal regulation of Wnt/β-catenin signaling is required for normal RPE development. Using chromatin immunoprecipitation (ChIP) and reporter gene assays we provide evidence that Otx2 and RPE-specific isoform of Mitf, Mitf-H, are direct transcriptional targets of Wnt/β-catenin signaling. Combined, our data suggest that Wnt/β-catenin signaling plays an essential role in development of RPE by maintaining or inducing expression of Mitf and Otx2.

    © 2009 Elsevier Inc. All rights reserved.


    The first indication of the vertebrate eye development is evagination of the diencephalon towards the surface ectoderm to form the optic vesicle (Chow and Lang, 2001). Lens-competent head ectoderm responds to signals from the optic vesicle, which induces columnar thickening of the surface epithelium to form the lens placode (Grainger et al., 1997). As the optic vesicle comes into contact with the surface ectoderm, it becomes partitioned into three terri- tories: a distal territory, a proximal territory and a dorsal territory, which give rise to the neural retina (NR), the optic stalk and the retinal pigment epithelium (RPE), respectively. Coordinated invagi- nation of the optic vesicle and the lens placode leads to formation of the double-layered optic cup and the lens vesicle. The inner layer and the outer layer of the optic cup give rise to the NR and RPE, res- pectively. The process of the invagination generates the optic fissure that runs from the ventral-most region of the NR and along the ventral aspect of the optic stalk. The optic fissure gradually becomes closed and the NR is completely surrounded by the RPE. The transition part between the NR and the RPE called the ciliary margin gives rise to the ciliary body and the iris (Bharti et al., 2006; Chow and Lang, 2001; Martinez-Morales et al., 2004).

    Although little is known about the RPE development, several transcription factors have been shown to be involved in the process.

    l rights reserved.

    Mitf, Otx1, and Otx2 are essential for the RPE development, while Chx10 prevents RPE development in the presumptive NR (Horsford et al., 2005; Martinez-Morales et al., 2004; Rowan et al., 2004). Mitf encodes a member of the basic helix–loop–helix leucine zipper family of transcription factors (Hodgkinson et al., 1993) and consists of nine isoforms with distinct amino-termini (Hallsson et al., 2007; Steingrimsson et al., 2004). Each isoform shows a unique expression pattern (Goding, 2000; Steingrimsson et al., 2004). For example,Mitf- A, -J, -H and -D are all expressed in the RPE, whereas expression of Mitf-M is restricted to the neural crest-derived melanocytes (Amae et al., 1998; Bharti et al., 2008; Hershey and Fisher, 2005; Takeda et al., 2002). Mitf regulates pigment cell-specific transcription of genes en- codingmelanogenic enzymes such as tyrosinase (Tyr), and tyrosinase- related protein 1 and 2 (Aksan and Goding, 1998; Hemesath et al., 1994; Yasumoto et al., 1994, 1997). During the vertebrate eye development Mitf is expressed in the entire optic vesicle, whereas later the expression is restricted to the RPE, the ciliary body and the iris (Baumer et al., 2003; Horsford et al., 2005; Nguyen and Arnheiter, 2000). The RPE of Mitf null mutants loses the expression of RPE- specific genes and transdifferentiates into the NR (Nguyen and Arnheiter, 2000). Otx1 and Otx2 encode members of the bicoid sub- family of homeodomain-containing transcription factors (Simeone et al., 1992). Similarly as Mitf, Otx1 and Otx2 are expressed in the entire optic vesicle and later expression is restricted to the presumptive RPE (Baumer et al., 2003; Martinez-Morales et al., 2001). Otx2 cooperates with Mitf to regulate expression of melanogenic enzymes (Martinez- Morales et al., 2003). Otx1 and 2 double-deficient mice show severe

  • 32 N. Fujimura et al. / Developmental Biology 334 (2009) 31–45

    ocular malformation in the lens, the NR, the optic stalk and the RPE. Notably, the presumptive RPE loses expression ofMitf and gives rise to the NR-like tissue (Martinez-Morales et al., 2001). The Chx10 gene encodes a member of paired-type homeodomain-containing tran- scription factor (Burmeister et al., 1996). Chx10 is expressed in the distal optic vesicle and at later stages restricted to the NR progenitor cells (Baumer et al., 2003; Burmeister et al., 1996; Chen and Cepko, 2000; Rowan et al., 2004). Chx10 represses expression of photo- receptor genes such as rod arrestin (Dorval et al., 2006). Chx10 null mutant mice show expansion of the peripheral RPE into NR and ectopic expression of Mitf in the entire NR (Horsford et al., 2005). Furthermore, misexpression of Chx10 in the developing RPE in chick results in significant downregulation of Mitf and tyrosinase-related protein 2, although transdifferentiation of the RPE does not occur (Rowan et al., 2004).

    It has been proposed that in addition to these transcription factors, secretedmolecules from the extraocular mesenchyme are required for RPE development to inhibit the NR development in the presumptive RPE (Fuhrmann et al., 2000). In the absence of the extraocular mesenchyme, explanted chick optic vesicles show downregulation of RPE-specific genes and ectopic expression of NR-specific genes. Activin A, a member of the TGFβ superfamily, can substitute for the extraocular mesenchyme (Fuhrmann et al., 2000). Bone morphoge- netic proteins (BMP), other members of the TGFβ superfamily, are essential for RPE development in chick embryos. The presumptive NR develops into the RPE by overexpression of BMPs, while inhibition of BMP results in abrogation of RPE development and in the induction of expression of the NR-specific genes (Muller et al., 2007).

    In multi-cellular organisms the Wnt signaling pathway repre- sents one of the key mechanisms controlling cell-fate decisions during embryonic development and also in adult tissues (Klaus and Birchmeier, 2008). The signaling is initiated by the interaction of extracellular Wnt ligands with the transmembrane Frizzled/LRP receptor complex. The activation of the receptor results in the stabi- lization of β-catenin, a key mediator of canonical Wnt signaling. The protein accumulates both in the cytoplasm and the nucleus, with the nuclear form able to act as a coactivator of Tcf/Lef transcription factors (Logan and Nusse, 2004). During eye development the Wnt pathway has been implicated in the formationof RPE. Several components of the canonical, i.e. β-catenin-dependent, Wnt signaling pathway, including Wnt2b, are expressed in the presumptive avian or mammalian RPE (Fuhrmann et al., 2000; Cho and Cepko, 2006; Jasoni et al., 1999; Jin et al., 2002; Liu et al., 2003; Zakin et al., 1998). In the mouse, Wnt/β- catenin signaling is highly active in the developing RPE at the stage of the optic cup formation and its activity is subsequently restricted to the ciliary margin (Kreslova et al., 2007; Liu et al., 2006, 2007; Maretto et al., 2003; Miller et al., 2006; Zhou et al., 2008). Moreover, recent studies have shown that Wnt/β-catenin-mediated signals are essen- tial for the ciliary margin development (Cho and Cepko, 2006; Kubo et al., 2003; Liu et al., 2007). Aberrant activation of the Wnt pathway in the peripheral NR leads to the expansion of the ciliary margin at the expense of the NR; on the contrary, conditional inactivation of the signaling attenuates the ciliary margin development (Liu et al., 2007). Interestingly, β-catenin is a polypeptide with dual roles and besides Wnt signaling also participates in cell adhesion (Grigoryan et al., 2008).β-catenin directly binds to the cytoplasmic tail of cadherins and associates withα-catenin, which links cadherin/catenin complexes to the actin cytoskeleton (Perez-Moreno et al., 2003). Although condi- tional knockout of β-catenin results in a fa